The present invention relates to quinolinium- and pyridinium-based fluorescent dyes, and to their use in the staining of proteins in solution, in gels and on solid supports.
Structures of formula II are disclosed in WO 96/36882, which discloses unsubstituted compounds (R1=R3=H) and the corresponding 2- and 2,6-substituted anilines, and those where the olefinic bond is optionally substituted (R4, R5=H, alkyl or phenyl), having fluorescence properties. A specific example of such a compound is shown in formula III.
Structures of formula IV are also disclosed in WO 96/36882, which discloses unsubstituted compounds (R1=R3=H) and the 2- and 2,6-substituted anilines, having fluorescence properties. A specific example of such a compound is shown in formula V. 
Citation of a reference herein shall not be construed as indicating that such reference is prior art to the present invention.
In a first aspect, the present invention relates to compounds of formula I 
wherein
R1 is (C1-C6) straight or branched chain alkyl, halogen or xe2x80x94CF3;
either R2a and R2b are independently a lipophilic group or H, R2a and R2b not simultaneously being H, or R2a and R2b are taken together and form a morpholinyl, piperidinyl or pyrrolidinyl ring;
R3 is H or (C1-C6) straight or branched chain alkyl; and
either R4and R5 are both H, or R4 and R5 taken together are xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94,
the aromatic rings A and B, the xe2x80x94(CH2)4-5xe2x80x94 group, and the xe2x80x94C(H)xe2x95x90C(R3)xe2x80x94 group being optionally substituted with one or more xe2x80x94OH, (C1-C6) straight or branched chain alkoxyl, halogen, (C1-C6) straight or branched chain haloalkyl, pyridyl, thiophenyl, furyl, and phenyl, the phenyl being optionally substituted with one or more xe2x80x94OH, (C1-C6) straight or branched chain alkyl or (C1-C6) straight or branched chain alkoxyl.
In a second aspect, the present invention relates to compounds of formula I wherein
R1 is H, (C1-C6) straight or branched chain alkyl, halogen or xe2x80x94CF3;
R2a and R2b are taken together to form xe2x80x94(CH2)2xe2x80x94NR6xe2x80x94(CH2)2xe2x80x94, wherein R6 is (C1-C12) straight or branched chain alkyl, (C1-C12) straight or branched chain alkylcarbonyl or (C1-C12) straight or branched chain alkylsulphonyl;
R3 is H; and
either R4and R5 are both H, or R4and R5 are taken together and form xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94,
the aromatic rings A and B, the xe2x80x94(CH2)4-5xe2x80x94 group, and the xe2x80x94C(H)xe2x95x90C(R3)xe2x80x94 group being optionally substituted with one or more xe2x80x94OH, (C1-C6) straight or branched chain alkoxyl, halogen, (C1-C6) straight or branched chain haloalkyl, pyridyl, thiophenyl, furyl, and phenyl, the phenyl being optionally substituted with one or more xe2x80x94OH, (C1-C6) straight or branched chain alkyl or (C1-C6) straight or branched chain alkoxyl.
In a third aspect, the present invention relates to compounds of formula I wherein
R1 is H, (C1-C6) straight or branched chain alkyl, halogen, or xe2x80x94CF3;
either R2a and R2b are independently a lipophilic group or H, R2a and R2b not simultaneously being H, or R2a and R2b are taken together and form a morpholinyl, piperidinyl, pyrrolidinyl, or piperazinyl ring, wherein the piperazinyl ring is optionally substituted with (C1-C12) straight or branched chain alkyl, (C1-C12) straight or branched chain alkylcarbonyl or (C1-C12) straight or branched chain alkylsulphonyl;
R3 is xe2x80x94CN, CONH2, xe2x80x94COOH, or xe2x80x94COOR, wherein R is (C1-C6) straight or branched chain alkyl or (C1-C10) straight or branched chain aralkyl, the xe2x80x94CONH2 group being optionally substituted with one or two (C1-C6) alkyl groups or one or two (C1-C10) aralkyl groups; and
either R4 and R5 are both H, or R4and R5 are taken together and form xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94,
the aromatic rings A and B, the xe2x80x94(CH2)4-5xe2x80x94 group, and the xe2x80x94C(H)xe2x95x90C(R3)xe2x80x94 group being optionally substituted with one or more xe2x80x94OH, (C1-C6) straight or branched chain alkoxyl, halogen, (C1-C6) straight or branched chain haloalkyl, pyridyl, thiophenyl, furyl, and phenyl, the phenyl being optionally substituted with one or more xe2x80x94OH, (C1-C6) straight or branched chain alkyl or (C1-C6) straight or branched chain alkoxyl.
The present invention may be understood more fully by reference to the detailed description and illustrative examples which are intended to exemplify non-limiting embodiments of the invention.
As used herein, halogen refers to F, Cl, Br or I.
As used herein, (C1-C6) straight or branched alkyl includes but is not limited to methyl, ethyl, n-propyl, propan-1-yl, propan-2-yl, cyclopropan-1-yl, n-butyl, butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, 3-methyl-n-butyl, n-pentyl, pentan-1-yl, pentan-2-yl, cyclopentan-1-yl, n-hexyl, hexan-1-yl, hexan-2-yl, and cyclohexan-1-yl.
As used herein (C1-C6) straight or branched chain alkoxy includes but is not limited to 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 4-hydroxybutyl, 1-hydroxybutyl, and 6-hydroxyhexyl.
As used herein, a lipophilic group refers to (C1-C20) straight or branched chain alkyl or (C1-C20) straight or branched chain aralkyl.
As used herein, (C1-C20) straight or branched chain alkyl includes but is not limited to methyl, ethyl, n-propyl, propan-1-yl, propan-2-yl, cyclopropan-1-yl, n-butyl, butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, 3-methyl-n-butyl, n-pentyl, pentan-1-yl, pentan-2-yl, cyclopentan-1-yl, n-hexyl, hexan-1-yl, hexan-2-yl, and cyclohexan-1-yl, n-octyl, n-decyl, n-hexadecyl, n-octadecyl, and n-eicosyl.
As used herein, aryl refers to phenyl, naphthyl, or anthracenyl.
As used herein, (C1-C6) straight or branched chain haloalkyl refers to an alkyl moiety, such as one of those listed above, having at least one halogen attached, including, but not limited to, xe2x80x94CF3, xe2x80x94CH2F, xe2x80x94CHF2, xe2x80x94CH2I, xe2x80x94CH2Br, xe2x80x94CH2Cl, xe2x80x94CHCl2, xe2x80x94CCl3, xe2x80x94CH2CHClCH3, xe2x80x94CH2C(CH3)(CH2Br), xe2x80x94CH2CH2CH2CH2I, xe2x80x94C(CH3)(CH3)(CH2F), and xe2x80x94CH2CHClCH2CHClCH2CH3. Preferably, the haloalkyl group is xe2x80x94CF3.
As used herein, (C1-C12) straight or branched chain alkylcarbonyl refers to an alkyl moiety, such as those listed above, having one or more Cxe2x95x90O groups attached.
As used herein, (C1-C12) straight or branched chain alkylsulphonyl refers to an alkyl moiety, such as those listed above, having one or more xe2x80x94SO2xe2x80x94 groups attached.
One skilled in the art will readily appreciate that when R4and R5 taken together are xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94, aromatic ring B and xe2x80x94CHxe2x95x90CHxe2x80x94CHxe2x95x90CHxe2x80x94 generate a quinoline system.
The compounds of formula I have been found to display fluorescence properties superior to those of the prior art. Thus, in particular, certain compounds of the invention display much increased brightness over the compounds of formulae III and V when they are bound to protein (e.g. bovine serum albumin) and when sodium dodecyl sulphate is present in an amount below its critical micelle concentration. Furthermore, certain compounds of this invention display greater differences in the fluorescence intensities of their protein-bound and unbound states when compared to the compounds of formulae III and V.
Without being bound by any particular theory, Applicants believe that the compounds of formula I have properties superior to those described in WO 96/36882. In general terms, other substituents (both those shown in formula I, and also those not shown) may be less critical; thus, each of the aromatic rings, the xe2x80x94(CH2)4-5xe2x80x94 group and the conjugating Cxe2x95x90C link may carry other/further substituents that do not substantially affect the fluorescence intensity of the compounds (e.g. compounds have at least 50%, and preferably at least 75%, of the intensity of any compound illustrated herein under the same conditions). Examples of substituents are disclosed in WO 96/36882, the contents of which are incorporated herein by reference.
Preferred compounds of formula I, of the first aspect of the invention, include:
Quinolinium, 4-[2-[4-(dipentylamino)-32-chlorophenyl]ethenyl]-1(sulfobutyl)-, inner salt;
Quinolinium, 4-[2-[4-(dipentylamino)-32-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-, inner salt;
Quinolinium, 4-[2-[4-(dipentylamino)-32-methylphenyl]ethenyl]-1-(sulfobutyl)-, inner salt;
Quinolinium, 4-[2-[4-(decylamino)-32-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-, inner salt; and
Pyridinium, 4-[2-[4-(dipentylamino)-32-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-, inner salt.
Particularly preferred compounds of formula I, of the first aspect of the invention, are:
Quinolinium, 4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-, inner salt;
Quinolinium, 4-[2-[4-(dipentylamino)-3-chlorophenyl]ethenyl]-1-(sulfobutyl)-, inner salt;
Quinolinium, 4-[2-[4-(decylamino)-3-trifluoromethylphenyl]ethenyl]-1-(sulfobutyl)-, inner salt; and
Pyridinium, 4-[2-[4-(dipentylamino)-3-trifluoromethylphenyl]ethenyl]1
(sulfobutyl)-, inner salt.
Preferred compounds of formula I, of the second aspect of the invention, include:
Quinolinium, 4-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner salt;
Quinolinium, 4-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner salt;
Pyridinium,4-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-inner salt;
Pyridinium, 4-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1(sulfobutyl)-, inner salt;
Pyridinium,2-[2-[4-[(1-oxooctyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-inner salt; and
Pyridinium, 2-[2-[4-[(1-oxodecyl)piperazinyl]phenyl]ethenyl]-1-(sulfobutyl)-, inner salt.
Preferred compounds of formula I, of the third aspect of the invention, include:
Pyridinium, 4-[2-[4-(dibutylamino)phenyl]-1-cyanoethenyl]-1-(sulfobutyl)-, inner salt; and
Pyridinium, 4-[2-[4-(dihexylamino)phenyl]-1-cyanoethenyl]-1-(sulfobutyl)-, inner salt.
Compounds of formula I may be prepared from starting materials that are known by procedures known to those of ordinary skill in the art. By way of illustration, a synthesis of the compounds of formula I is shown in Scheme A, below. Compounds of formula Al are disclosed in EP-A-0548798. Non-limiting examples of the reactions involved may be found in, or based on, the Examples below. An illustrative synthesis of the compounds of formula I wherein R3 is an electron withdrawing substituent as defined in the third aspect of the invention is shown in Scheme B, below. 
Compounds of the invention have utility in the visualisation of proteins following one-dimensional or 2-dimensional polyacrylamide gel electrophoresis (xe2x80x9cPAGExe2x80x9d).
Post-electrophoresis staining with such compounds allows the detection of proteins, typically down to the level of 2-20 femtomoles. Procedures for this purpose are known to those of ordinary skill in the art. A preferred system for 2-dimensional electrophoresis, detection, identification and isolation of proteins in biological samples is described in U.S. application Ser. No. 08/980,574, filed Dec. 1, 1997 (published as WO 98/23950), which is incorporated herein by reference in its entirety and which sets forth a preferred protocol at pages 29-35. In a preferred embodiment, a biological sample is treated, prior to electrophoresis, to enrich biomolecules (e.g. proteins) of interest or to deplete biomolecules (e.g. proteins) that are not of interest, as described in International Application No. PCT/GB99/01742, filed Jun. 1, 1999, which is incorporated by reference in its entirety, with particular reference to pages 3 and 6.
For example, a sample (e.g. plasma or serum) may be processed to deplete or remove one or more proteins such as albumin, haptoglobin, transferrin, alpha-1-antitrypsin, alpha-2-macroglobulin and immunoglobulin G (IgG) by performing affinity chromatography whereby the sample is passed through a series of columns containing immobilized antibodies for selective removal of albumin, haptoglobin, transferrin, alpha-1-antitrypsin and alpha-2-macroglobulin, and containing protein G for selective removal of IgG. In one such embodiment, two affinity columns in a tandem assembly are prepared by coupling antibodies to protein-G sepharose contained in 1 ml columns (Protein G-sepharose xe2x80x9cHi-Trapxe2x80x9d columns, Pharmacia Cat. No. 17-0404-01) by circulating the following solutions sequentially through the columns: (1) Dulbecco""s Phosphate Buffered Saline (Gibco BRL Cat. No.14190-094); (2) concentrated antibody solution; (3) 200 mM sodium carbonate buffer, pH 8.35; (4) cross-linking solution (200 mM sodium carbonate buffer, pH 8.35, 20 mM dimethylpimelimidate); and (5) 500 mM ethanolamine, 500 mM NaCl. A third (underivatized) protein G Hi-Trap column is then attached in series with and following the tandem antibody column assembly. The chromatographic procedure may be automated using an Akta Fast Protein Liquid Chromatography (FPLC) System such that a series of up to seven runs can be performed sequentially. The samples are passed through the series of 3 Hi-Trap columns, in which the affinity chromatography media selectively bind the above proteins, thereby depleting or removing them from the sample. Typically fractions (3 ml per tube) are collected of unbound material (xe2x80x9cflowthrough fractionsxe2x80x9d) that elutes through the column during column loading and washing, and of bound proteins (xe2x80x9cbound/eluted fractionsxe2x80x9d) that are eluted by step elution with Immunopure Gentle Ag/Ab Elution Buffer (Pierce Cat. No. 21013). The eluate containing unbound material is collected in fractions which are pooled, desalted and concentrated by centrifugal ultrafiltration, and stored to await further analysis.
A preferred scanner for detecting fluorescently labeled proteins is described in WO 96/36882 and in the Ph.D. thesis of David A. Basiji, entitled xe2x80x9cDevelopment of a High-throughput Fluorescence Scanner Employing Internal Reflection Optics and Phase-sensitive Detection (Total Internal Reflection, Electrophoresis)xe2x80x9d, University of Washington (1997), Volume 58/12-B of Dissertation Abstracts International, page 6686, the contents of each of which are incorporated herein by reference. This document describes a new image scanner designed specifically for automated, integrated operation at high speeds. The scanner can image gels that have been stained with fluorescent dyes or silver stains, as well as storage phosphor screens. The scanner incorporates a phase-sensitive detection system for discriminating modulated fluorescence from baseline noise due to laser scatter or homogeneous fluorescence. This capability increases the sensitivity of the instrument by an order of magnitude or more compared to conventional fluorescence imaging systems. The increased sensitivity reduces the sample-preparation load on the upstream instruments while the enhanced image quality simplifies image analysis downstream in the process.